In vitro Germination,Callus Induction and Primary Haustorium Organogenesis in the Parasitic Plant Cynomorium songaricum

Xin YUE, Gui-Lin CHEN

Bulletin of Botanical Research 2020, 40 (6): 846-854. DOI: 10.7525/j.issn.1673-5102.2020.06.007

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Cynomorium songaricum Rupr. is an obligate parasitic plant widely used as traditional Chinese medicine and Mongolian medicine. Here， we firstly describe protocols for in vitro germination， callus induction and haustorium organogenesis in C.songaricum Rupr. In this study， adequate concentrations of gibberellic acid（GA₃），_{in combination with other plant growth regulators in the medium， promoted embryo development and germination of C.songaricum seeds. The highest callus induction rate from seeds（13.7%） after a 40 d incubation was obtained with B₅ medium adding 2，4-dichlorophenoxyacetic acid（2，4-D； 1.0 mg·L^-1）， kinetin（KT；0.5 mg·L^-1） and GA₃（1.0 mg·L^-1. This resulted in callus_{formation in 13.7%±3.1% of seeds. Addition of 2，4-D（0.5 mg·L^-1） and KT（0.25 mg·L^-1） yielded highest haustorium organogenesis from calluses. Some primary haustorium branched to form adventitious roots of 3-4 cm in length. Subsequently， the tip of each adventitious root formed a nascent primary haustorium， which was then branched out into adventitious roots. The role of auxins（2，4-D in this study） in the formation of primary haustorium and adventitious roots from seed callus in C.songaricum was also discussed.}}

Fig.1 Development of C.songaricum embryo in different periods of cultivation
Seeds cultured for 0, 20, 30 and 40 d were examined.The embryos were obtained by crushing the seeds with another microscope slide, transferred to the formalin-aceto-alcohol(FAA) stationary liquid medium for 24 h, and observed with bright ?eld illumination(Fig.1A,C and D). The embryo was stained with a fluorochrome and observed with a fluorescence objective lens(Fig.1B)

The tiny embryo of C.songaricum appeared as a multicellular spherical proembryo with no germ cell differentiation， with a radicle or cotyledon found close to the micropylar end（Fig.1A）. Embryo cells varied in size， with larger and smaller ones located near the chalazal and micropylar ends， respectively（Fig.1B）. The cells were polyhedral in shape， tightly packed and contained large nuclei.

Seeds cultured for 0, 20, 30 and 40 d were examined.The embryos were obtained by crushing the seeds with another microscope slide, transferred to the formalin-aceto-alcohol(FAA) stationary liquid medium for 24 h, and observed with bright ?eld illumination(Fig.1A,C and D). The embryo was stained with a fluorochrome and observed with a fluorescence objective lens(Fig.1B)

During culture， the embryos initially became differentiated at two poles（Fig.1C）. This was followed by unipolar development during the process of germination（Fig.1D）. The level of embryonic development and differentiation of mature C.songaricum seeds was similar to that of other parasitic angiosperms. The present findings support these previous observations.